Tetrahydrobiopterin (BH4) is the cofactor for tyrosine hydroxylase and tyrosine hydroxylase and tryptophan hydroxylase, which are the initial and rate-limiting enzymes in catecholamine and serotonin synthesis. BH4 is an important regulator of catecholamine synthesis, since BH4 administration increases brain dopamine synthesis, whereas inhibition of BH4 synthesis leads to catecholamine deficits. In BH4 biosynthesis, GTP cyclohydrolase is the initial enzyme, whereas sepiapterin reductase catalyzes the final reaction. In rodent, GTP cyclohydrolase may be the main rate-controlling enzyme in BH4 synthesis; in humans, other enzymes such as sepiapterin reductase may contribute to regulating BH4 production, though further study is required. In rat adrenal medulla, catecholamine depletion elevates tyrosine hydroxylase and GTP cyclohydrolase activities and raises BH4 levels, indicating that "coordinate regulation" of these enzymes may occur. Coordinate regulation refers to a coordinated response in tyrosine hydroxylase, GTP cyclohydrolase, and sepiapterin reductase gene expression when BH4 and catecholamine metabolism is altered. The elevation of adrenal BH4 due to catecholamine depletion is thought o be crucial for maintaining increased tyrosine hydroxylation in vivo when less catalytically-active tyrosine hydroxylase molecules are synthesized at early stages. In brain, preliminary data indicates that kainic acid lesions of non-dopamine cells in striatum elevates striatal tyrosine hydroxylase and CTP cyclohydrolase activities. In newborns with atypical phenylketonuria (PKU), genetic defects in any one of the several BH4 biosynthetic enzymes lead to BH4 deficiency in liver and brain. This results in a biogenic amine deficiency in brain and neurological impairment. Since many other systems requiring biogenic amine synthesis function normally in these patients, atypical PKU may be caused by tissue-specific regulatory mutations of BH4 metabolism. Mutations of BH4 biosynthesis may explain altered BH4 and catecholamine deficits observed in normal aging, Alzheimer's and Parkinson's diseases, and familial dystonia. Further understanding of BH4 biosynthesis will be obtained by studying coordinate regulation of tyrosine hydroxylase, GTP cyclohydrolase, and sepiapterin reductase gene expression in animal models of aging and neuro-degeneration, and following drug treatments influencing BH4 and catecholamine synthesis in the adrenal medulla tumor cell line, pheochromocytoma (PC12) cells. As a substitute for animals, PC12 cells can be exposed to a wider variety of drugs affecting adrenal BH4 and catecholamine metabolism under well-controlled tissue culture conditions. Thus, coordinate regulation at the level of BH4 and catecholamine gene expression will be studied in rat adrenal medulla, PC12 cells, and for the first time in brain. Expression of these enzymes will be monitored by measuring tissue MRNAS (Northern blots or ribonuclease protection assays), amounts of enzymes (Western blots), enzyme activities, and end-products of biosynthesis (BH4 and catecholamines) following treatments with: 1) inhibitors of each of these enzymes; 2)activators of BH4 and catecholamine synthesis, and 3) and kainic acid in striatum. Kainic acid will also be used to examine aging effects on gene expression in surviving nigrostriatal dopamine neurons following neurotoxic damage. These results will provide direction for studies examining human BH4 and catecholamine-related gene expression, and the existence of neuropsychiatric diseases related to regulatory mutations of BH4 biosynthesis. Human studies can be accomplished once the human BH4-related gene probes are cloned, which is ongoing in the PI's laboratory.